Nitric oxide (NO)reduces blood pressure by dilating blood vessels and promoting renal salt excretion. Thick ascending limbs absorb 30% of the filtered NaCI. Inappropriate salt retention by this segment leads to hypertension. We reported that NO produced by NO synthase (NOS) 3 in the thick ascending limb acts as an autacoid to inhibit transport. Flow augments NOS 3 activity via mechano-sensitive channels in endothelial cells. Our preliminary data show that luminal flow activates NOS 3 in thick ascending limbs and this requires translocation of the enzyme to the luminal membrane. However, it is not known how luminal flow regulates NOS 3 activity in this segment or how flow-induced NO affects NaCI reabsorption. We hypothesize that increasing luminal flow in thick ascending limbs stimulates NO production by NOS 3 via activation of mechano-sensitive TRPV4 channels, release of ATP and activation of Akt.The NO thus produced blunts NaCI reabsorption. Defects in the response to flow-stimulated NO production enhance salt retention and promote salt-sensitive hypertension. This hypothesis will be tested in 4 aims. Aim I. Hypothesis: Increasing luminal flow causes translocation and activation of NOS 3, stimulating NO production by the thick ascending limb due to increased shear stress. Aim II. Hypothesis: Luminal flow enhances NOS 3 activity by activating TRPV 4 channels and causing local increases in intracellular Ca. Aim III.Hypothesis: TRPV 4 activation by luminal flow induces release of ATP, which binds to purinergic 2 receptors and activates phosphatidylinositol 3 kinase, Akt and NOS 3. Aim IV: Hypothesis: Flow-stimulated NO production inhibits Na reabsorption by thick ascending limbs via activation of cGMP-stimulated phosphodiesterase 2 and reductions in cAMP. Defects in the response to flow-enhanced NO production promote salt retention and salt-sensitive hypertension. We will study how flow stimulates NO in perfused tubules and cultured cells using physiological, imaging, molecular and biochemical methods. Project 1 relates to the central theme since it will study renal NOS 3 regulation and the effects of NO on salt absorption. Information from Project 1 will be integrated with that from all other projects. It will use all of the cores. These will be the first studies to define how flow activates NOS 3, its effects on Na reabsorption by the thick ascending limb and whether these effects are blunted in salt-sensitive hypertension. This information may lead to new therapeutic targets for the treatment of hypertension.